The present invention relates generally to methods for transforming plant cells and, more particularly, to methods for identifying plants in which a target DNA sequence has been modified by homologous recombination.
Efficient methods for identifying plant cells in which homologous recombination has occurred is extremely useful in developing new plant varieties with targeted replacement or alteration of endogenous genes. Activation of a reporter gene activity due to homologous recombination between two non-allelic mutant copies of a reporter gene or between two overlapping deletions of a reporter gene have been reported in the literature (see, Assaad, et al., Genetics, 132:553-566 (1992); Tovar, et al., Plant Cell, 4:319-332 (1992); Baur, et al., Mol. Cell. Biol., 10:492-500 (1990); Chiurazzi, et al., Plant Cell, 8(11):2057-2066 (1996); Hrouda, et al., Mol. Gen. Genet. 243:106-111 (1994); Lebel, et al., Proc. Natl. Acad. Sci. USA, 90:422-426 (1993); Lyznik, et al., Mol. Gen. Genet. 230:209-218 (1991); Offringa, et al. Proc. Natl. Acad. Sci. USA, 90:7346-7350 (1993); Offringa, et al., EMBO J, 9:3077-3084 (1990); Swoboda, et al., EMBO J, 13:484-489 (1994); Risseeuw, et al., Plant J, 7:109-119 (1995); Puchta, et al., Proc. Natl. Acad. Sci. USA 93:5055-5060 (1996); Puchta, et al., Plant Mol. Biol. 28:281-292 (1995); Puchta, et al., Nuc. Acids Res., 21:5034-5040 (1993); Puchta, et al., Mol. Cell. Biol., 12:3372-3379 (1992); Puchta, et al., Nuc. Acids Res. 19:2693-2700 (1991); Puchta, et al., Mol. Gen. Genet., 230:1-7 (1991); Paszkowski, et al., EMBO J, 7:4021-4026 (1988); Peterhans, et al., EMBO J., 9:3437-3446 (1990)). However, these reports are limited to identifying recombination events within genes which themselves had some inherent xe2x80x9creporter gene activityxe2x80x9d (e.g. drug resistance or conversion of a calorimetric substrate). In most cases, the homologous recombination events were non-reciprocal (unidirectional) homology dependent events, referred to as gene conversion events.
Several reports in animal gene targeting experiments use a dominant selectable reporter gene as part of a xcex94gene X::reporter gene fusion to select for homologous recombination events in vivo (Buerstedde, et al., Cell, 67:179-88 (1991); Jasin, et al., Genes and Devel., 2:1353-63 (1988); Sedivy, et al., Proc. Natl. Acad. Sci. USA, 86:227-31 (1989)). In all cases, the reporter genes used imparted resistance to a drug that was used as a dominant positive selection for those cells that underwent the specific homologous recombination between the xcex94gene X::reporter gene and the endogenous gene. A dominant selection approach can be problematic because either an insufficient level of the drug resistance trait or a tissue specific gene X expression pattern of the drug resistance trait may result in an inability to survive the drug selection during cell culture. The same concerns regarding the level and tissue specific expression pattern of the recombinant gene Xxe2x80x2/X::reporter gene apply to the situations of using a dominant selectable reporter gene on a whole organism-based screen.
Although progress has been made, efficient screening methods for detecting homologous recombinants, particularly in plant cells is needed in the art. The present invention provides these and other advantages.
The present invention provides compositions and methods which use a reporter sequence to identify homologous recombinants in a large population of transformed plants or plant cells. The methods of the invention involve contacting plant cells with a nucleic acid molecule comprising a fusion polynucleotide sequence comprising a polypeptide sequence of interest linked to a reporter sequence, wherein the nucleic acid molecule lacks sequences necessary for expression of the fusion polynucleotide sequence gene product in a cell. The fusion polynucleotide sequence gene product is then detected in the plant cells, thereby identifying plant cells in which homologous recombination has occurred.
The means by which the fusion polynucleotide is introduced into the cell is not critical. Typically, the polynucleotide is introduced using a T-DNA vector. In some embodiments, plants are regenerated from the plant cells before the step of detecting the presence of the fusion sequence gene product.
The particular reporter sequence used in the methods is also not critical, for instance, non-selective markers such as luciferase can be used. In this case video imaging equipment is conveniently used to detect the luciferase.
The homologous recombination event can be used to alter endogenous gene in any number of ways. For instance, the recombination can result in gene conversion or may lead to inactivation of the endogenous gene. Alternatively, a recombinant allele derived from two related genes can be produced. The newly created recombinant allele will typically have a new activity as compared to either of the genes from which it was derived.
The invention also provides isolated nucleic acid molecule useful in the above methods, as well as plants produced by the methods.
The term xe2x80x9chomologous recombinationxe2x80x9d refers to the process of recombination between two nucleic acid molecules based on nucleic acid sequence similarity. The term embraces both reciprocal and nonreciprocal recombination (also referred to as gene conversion). In addition, the recombination can be the result of equivalent or non-equivalent cross-over events. Equivalent crossing over occurs between two equivalent sequences or chromosome regions, whereas nonequivalent crossing over occurs between identical (or substantially identical) segments of nonequivalent sequences or chromosome regions. Unequal crossing over typically results in gene duplications and deletions. For a description of the enzymes and mechanisms involved in homologous recombination see, Watson et al., Molecular Biology of the Gene pp 313-327, The Benjamin/Cummings Publishing Co. 4th ed. (1987).
The phrase xe2x80x9cnucleic acid sequencexe2x80x9d refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
A xe2x80x9cpromoterxe2x80x9d is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. As used herein, a xe2x80x9cplant promoterxe2x80x9d is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A xe2x80x9cconstitutivexe2x80x9d promoter is a promoter that is active under most environmental and developmental conditions. An xe2x80x9cinduciblexe2x80x9d promoter is a promoter that is active under environmental or developmental regulation. The term xe2x80x9coperably linkedxe2x80x9d refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
The term xe2x80x9cplantxe2x80x9d includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds and plant cells and their progeny. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is xe2x80x9cheterologous toxe2x80x9d an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.
A polynucleotide xe2x80x9cexogenous toxe2x80x9d an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as an R1 generation transgenic plant. Transgenic plants which arise from sexual cross or by selfing are descendants of such a plant.
Two nucleic acid sequences or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers and Miner, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A xe2x80x9ccomparison windowxe2x80x9d, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins and Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat""l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993). Generally, highly stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30 xc2x0 C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
In the present invention, genomic DNA or cDNA comprising FIE nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and at least one wash in 0.2xc3x97SSC at a temperature of at least about 50xc2x0 C., usually about 55xc2x0 C. to about 60xc2x0 C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.